EP1270732A1 - Transfektion von eukaryotischen Zellen mit linearen Polynukleotiden mittels Elektroporation - Google Patents

Transfektion von eukaryotischen Zellen mit linearen Polynukleotiden mittels Elektroporation Download PDF

Info

Publication number
EP1270732A1
EP1270732A1 EP01115076A EP01115076A EP1270732A1 EP 1270732 A1 EP1270732 A1 EP 1270732A1 EP 01115076 A EP01115076 A EP 01115076A EP 01115076 A EP01115076 A EP 01115076A EP 1270732 A1 EP1270732 A1 EP 1270732A1
Authority
EP
European Patent Office
Prior art keywords
cells
mrna
electroporation
transfected
egfp
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP01115076A
Other languages
English (en)
French (fr)
Inventor
Gerold Prof. Dr. Schuler
Zwi N. Prof. Berneman
Viggo F.I. Dr. Van Tendeloo
Peter Ponsaerts
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Nv Antwerpes Innovatiecentrum
Schuler Gerold
Original Assignee
Nv Antwerpes Innovatiecentrum
Schuler Gerold
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Nv Antwerpes Innovatiecentrum, Schuler Gerold filed Critical Nv Antwerpes Innovatiecentrum
Priority to EP01115076A priority Critical patent/EP1270732A1/de
Priority to EP10185221.8A priority patent/EP2308987B1/de
Priority to KR10-2003-7016741A priority patent/KR20040025690A/ko
Priority to PCT/EP2002/006897 priority patent/WO2003000907A2/en
Priority to CA2451389A priority patent/CA2451389C/en
Priority to CN028163621A priority patent/CN1701121B/zh
Priority to BR0210936-0A priority patent/BR0210936A/pt
Priority to EP02748810.5A priority patent/EP1397500B1/de
Priority to JP2003507290A priority patent/JP2004536598A/ja
Publication of EP1270732A1 publication Critical patent/EP1270732A1/de
Priority to AU2008202507A priority patent/AU2008202507B2/en
Priority to JP2010265545A priority patent/JP2011050395A/ja
Priority to AU2011201652A priority patent/AU2011201652C1/en
Withdrawn legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4615—Dendritic cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/462—Cellular immunotherapy characterized by the effect or the function of the cells
    • A61K39/4622—Antigen presenting cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/46449—Melanoma antigens
    • A61K39/464491—Melan-A/MART
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • the present invention provides an improved method for gene delivery in eukaryontic cells by electroporation, preferably in human hematopoietic cells, particular dendritic cells.
  • the method of the invention is superior to lipofection and passive pulsing of mRNA and to electroporation of plasmid cDNA for gene delivery, including tumor antigen loading of dendritic cells.
  • DC Dendritic cells
  • cytotoxic T lymphocytes TTL-mediated anti-tumor immune responses
  • CTL cytotoxic T lymphocytes
  • studies have documented viral transfer of cDNA encoding tumor-associated antigens to load DC for induction of TAA-specific cytotoxic T lymphocytes (CTL) (Dietz, A.B., Vuk, P.S., Blood, 91:392-398 (1998); Brossart, P. et al., J. Immunol., 158:3270-3276 (1997); Specht, J.M. et al., J. Exp. Med., 186:1213-1221 (1997)) nonviral gene delivery systems for DC-based vaccines would provide a more attractive approach with clinical perspectives since safety issues and immunogenicity of the vector are reduced to a minimum.
  • DC can be transfected to comparable levels as compared to transduction by recombinant viruses, such as poxviruses Kim, C. J. et al., J. Immunother., 20:276-286 (1997)) or adenoviruses (Dietz, A. B., Vuk, P.S., Blood, 91:392-398 (1998)), while circumventing the drawbacks of viral vectors (Jenne, L. et al., Gene Ther., 7:1575-1583 (2000); Jonuleit, H.
  • RNA has a short cellular half-life and lacks the potential to integrate into the host genome, thereby obviating safety concerns, e.g. insertional mutagenesis, in the context of clinical gene therapy trials (Lu, D. et al., Cancer Gene Ther., 1:245-252 (1994); Ying, H. et al., Nat. med., 5:823-827 (1999)).
  • Electroporation methods for the integration of naked polynucleic acids into "normal" cells are generally using the following reaction conditions (Van Tendeloo V.F.I et al., Gene Ther. 5:700-707 (1998); Van Tendeloo, V.F.I. et al., Gene Ther. 7:1431-1437 (2000); Van Bockstaele, D., Berneman, Z.N., Cytometry 41:31-35 (2000); Lurquin, P.F., Mol. Biotechnol. 7:5-35 (1997); Matthews, K.E. et al., Mol. Biotechnol., vol 48, Chapter 22, Ed.Nickoloff); Spencer, S.C., Biochem. and Biotechnol. 42:75-82 (1993)):
  • transfection efficiency in Mo-DC, 34-DC and 34-LC was at least 25, 6 and 3 times, respectively, more efficient as compared to plasmid DNA electroporation (Van Tendeloo, V.F.I. et al., Gene Ther., 5:700-707 (1998)), and also superior to previously described mRNA electroporation. Also, mRNA electroporation was superior to mRNA lipofection and passive pulsing. This increased transfection efficiency was translated in a superior biological effect in vitro, as confirmed by our CTL activation experiments, and could be used as a tool to investigate as to whether it results in a higher immunopotency in vivo (Porgador, A.
  • mRNA-transfected DC were able to efficiently process the introduced antigen and present antigenic epitopes in an MHC class I-restricted manner to a specific CD8 + TIL clone (Fig. 4).
  • antigen loading by mRNA electroporation was preferably performed prior to DC maturation in order to achieve the most optimal antigen presentation (Fig. 5), indicating the importance of the sequence of loading and DC maturation for future DC-based vaccine design Morse, M. A. et al., Cancer Res., 58:2965-2968 (1998)).
  • the present invention describes a method for high-efficiency non-viral transfection of Mo-DC as well as other types of dendritic cells (including CD34 + derived Langerhans cells and interstitial type DC by mRNA electroporation correlated with effective loading of tumor antigens into different types of human DC.
  • the efficiency of the method of the present invention was compared with other transfection methods, such as lipofection and passive pulsing of mRNA as well as cDNA electroporation, and found to be highly superior. Furthermore, the effect of DC maturation on loading efficiency was investigated. An electroporation-based mRNA transfection protocol was developed which is suitable for highly efficient antigen loading in Mo-DC, as well as in 34-DC and 34-LC. This technique proved to be superior to mRNA lipofection or passive mRNA pulsing in terms of loading efficiency and subsequent activation of an antigen-specific CD8 + CTL clone.
  • the present invention thus provides
  • DC-based tumor vaccines Designing effective strategies to load human dendritic cells (DC) with antigens such as tumor antigens) is a challenging approach for DC-based tumor vaccines. Also, the expression of other proteins (e.g., stimulatory or tolerogenic or apoptotic molecules) in DC by gene transfer might be desired, furthermore the introduction of antisense RNA by electroporation.
  • the present invention describes a cytoplasmic expression system based on mRNA electroporation to efficiently introduce genetic information into DC.
  • mRNA-electroporated DC retained their phenotype and maturational potential.
  • DC electroporated with mRNA encoding Melan-A strongly activated a Melan-A-specific cytotoxic T lymphocyte (CTL) clone in an HLA-restricted manner and were superior to mRNA-lipofected or - pulsed DC.
  • CTL cytotoxic T lymphocyte
  • Optimal stimulation of the CTL occurred when Mo-DC underwent maturation following mRNA transfection. Strikingly, a nonspecific stimulation of CTL was observed when DC were transfected with plasmid DNA.
  • Our data clearly demonstrate that Mo-DC electroporated with mRNA efficiently present functional antigenic peptides to cytotoxic T cells.
  • electroporation of mRNA encoding tumor antigens is a powerful technique to charge human dendritic cells with tumor antigens and could serve applications in future DC-based tumor vaccines.
  • Transfection of ready mature was less efficient when maturation stimuli such as TNF ⁇ + LPS were used.
  • the use of a certain generation methanol for Mo-DC including an optimized maturation stimulus allowed, however, also for efficient transfection such as tumor antigens of mature Mo-DC.
  • Figure 1 shows the flow cytometric analysis of transgene expression in K562 cells following EGFP mRNA electroporation.
  • Figure 2 shows the FCM analysis of transgene expression following EGFP mRNA transfection in different types of DC.
  • Figure 3 shows the phenotypical analysis and maturation potential of mRNA-electroporated DC.
  • FIG. 4 shows the mRNA-based antigen loading of Mo-DC.
  • the SK23-MEL melanoma cell line, HLA-A2 + Mo-DC pulsed with a Melan-A or irrelevant influenza peptide and HLA-A2-negative Mo-DC electroporated with Melan-A mRNA served as controls.
  • Antigen-presenting cells (indicated on the left of the graph) were co-incubated with a Melan-A specific CD8 + CTL clone to determine antigen loading eficiency, as reflected by IFN- ⁇ production of the CTL clone. Results are shown as mean ⁇ SD. * P ⁇ 0.05; EP, electroporation; lipo, lipofection; puls, passive pulsing.
  • Figure 5 shows the effect of DC maturation on tumor antigen presentation of mRNA-transfected Mo-DC.
  • IFN- ⁇ production by the CTL clone was measured after coculture with HLA-A2 + Mo-DC electroporated with Melan-A mRNA.
  • iMo-DC Mo-DC transfected at the immature stage and used as such;
  • Mo-DCa Mo-DC transfected at the mature stage after LPS+TNF- ⁇ stimulation;
  • Mo-DCb Mo-DC transfected at the immature stage, matured by LPS+TNF- ⁇ and then assayed for Melan-A-specific CTL clone stimulation.
  • Figure 6 shows the outcome of plasmid cDNA-based antigen loading of 34-LC.
  • Figure 7 shows the result of electroporation of immature monocyte-derived cells, in particular, the phenotype of dendritic cells 48 h after electroporation with GFP-RNA.
  • the numbers in the lower right part of the quadrant indicate the EGFP-positive DC, the numbers in the upper right part show the EGFP+/CD83+ and EGFP+/CD25+ DC, respectively.
  • Figure 8 shows the transfection efficiency of and kinetics of EGFP expression in dendritic cells following GFP-RNA-transfection using electroporation.
  • Figure 9 shows the results of EGFR RNA-transfection of monocyte-derived dendritic cells by electroporation.
  • Figure 10 EGFP RNA-transfection of mature monocyte-derived dendritic cells by electroporation.
  • Figure 11 FCM analysis of transgene expression in immature and mature DC after EGFP mRNA electroporation in non-frozen controls and after thawing of cryopreserved samples.
  • the dot plots show EGFP fluorescence on the x-axis and ethidium bromide staining on the y-axis. Analysis was performed on cells exhibiting a large forward scatter and large side scatter profile, in order to allow exclusion of contaminating autologous lymphocytes. Percentage of dead cells (upper left corner), viable EGFP+ cells (lower right corner) and viable EGFP- cells (lower left corner) is indicated based on the number of dots in the quadrant analysis.
  • A Dot plots show analysis of non-frozen iMo-DC 24 hours after mRNA electroporation (left), and of mRNA-electroporated iMo-DC 6 hours after thawing (middle) and 24 hours (right) after thawing.
  • B Dot plots show analysis of non-frozen mMo-DC 24 hours after mRNA electroporation (left),and of mRNA-electroporated mMo-DC 6 hours after thawing (middle) and 24 hours (right) after thawing.
  • EP electroporation.
  • Figure 12 Representative example of phenotypical analysis of non-frozen and frozen mRNA-electroporated immature and mature DC. Dot plots show FCM analysis of PE-labeled monoclonal antibodies directed against typical DC-markers including CD1a, HLA-DR, CD80 and CD86 (y-axis). As controls to set quadrants, isotype-matched antibodies and a PE-labeled monoclonal CD14 antibody was used. Analysis of DC markers was done on viable EGFP- cells in control samples and on viable EGFP+ cells in mRNA-electroporated DC as shown by the EGFP fluorescence on the x-axis.
  • E EGFP+ iMo-DC after mRNA electroporation on day 6 and stimulation for 48 hours with the maturation cocktail
  • F EGFP+ iMo-DC after mRNA electroporation on day 6 and culture for 24 hours with a maturation cocktail, cryopreservation, thawing and culture for 24 hours in presence of the maturation cocktail.
  • phenotyping was performed after 2 days of culture, with or without a frozen interval (that was not counted), following day 6 of the Mo-DC culture.
  • FIG. 13 Stimulatory capacity of cryopreserved mRNA-electroporated mature DC.
  • Cryopreserved matrix protein M1 mRNA-electroporated mature DC were used as stimulators for PBMC during a 6 day coculture.
  • Primed PBMC were then stimulated with T2 cells, pulsed with an MHC class I-restricted M1 immunodominant epitope, during a 6 hour coculture.
  • Antigen specific T cells in the primed PBMC culture were detected as shown by positive IFN- ⁇ production.
  • unpulsed T2 cells were used as stimulators and fresh PBMC as responders. Results are shown as mean ⁇ standard error.
  • RNA electroporation For the electroporation, the following parameters were most preferred: a 4 mm cuvette with 200 ⁇ l of cell suspension and we shock the cells using 300 volts and a capacitance of 150 ⁇ F (pulse time 8-10 ms). These are optimal parameters for both leukemic K562 cells and different types of DC, both progenitor- and monocyte-derived DC. In the optimization process, other parameters were also checked, e.g., by ranging the voltage and the capacitance, as well as the volume in the cuvette, resulting in shorter or longer pulse times. In summary the following parameters for efficiency and toxicity of RNA electroporation were analyzed:
  • RNA electroporation is the low voltage (range 100 V-450 V), combined with a low capacitance (150-300 ⁇ F) (which is in contrast to DNA settings, for which a high capacitance is required) and a low electroporation volume (200 ⁇ l) to increase cell concentration.
  • Electroporation and incubations are all performed at room temperature and cells are resuspended in serumfree buffer (e.g. IMDM, RPMI, Opti-MEM) or in optimized electroporation buffer Opti-Mix purchased from EquiBio, UK cat n# EKIT-E1).
  • serumfree buffer e.g. IMDM, RPMI, Opti-MEM
  • optimized electroporation buffer Opti-Mix purchased from EquiBio, UK cat n# EKIT-E1).
  • Our electroporator type is Easyject Plus (EquiBio) which only delivers exponential decay pulses.
  • a Gene Pulser II Biorad was used.
  • the preferred range of voltage is 100-500 V
  • the range of capacitance is 150-1500 ⁇ F
  • the range of pulse time is 5-40 ms.
  • IVT mRNA-based electroporation is a highly efficient and simple nonviral method to gene-modify human Mo-DC, 34-DC and 34-LC with tumor antigens.
  • the technique described in this study can serve applications in DC-based tumor vaccine development and in other gene transfer protocols requiring high-level short-term transgene expression in hematopoietic cells.
  • T2 cells (TAP-deficient, HLA-A2 + , TxB hybrid), EBV-LG2 (HLA-A2 - EBV-transformed B lymphocytes), and SK23-MEL (Melan-A + HLA-A2 + melanoma cell line) were kindly provided by Dr. Pierre Van der Bruggen (Ludwig institute for Cancer Research, Brussels, Belgium). K562 cells were obtained from the American Type Culture Collection (ATCC n° CCL-243, Rockville, MD, USA).
  • IMDM Iscove's medium
  • L-glutamine 2 mM
  • penicillin 100 U/ml
  • streptomycin 100 ⁇ g/ml
  • amphotericin B 1.25 ⁇ g/ml Fungizone
  • FCS 10% fetal calf serum
  • the CD8 + TIL 1235 clone recognizing the immunodominant HLA-A0201-restricted Melan-A 27-35 epitope was a kind gift of Dr. J. Wunderlich (NIH, Bethesda, USA) and was cultured as described earlier with minor modifications (Reeves, M. E. et al., Cancer Res., 56:5672-5677 (1996)).
  • the TIL clone was maintained in AIM-V medium (Gibco BRL) supplemented with 5% pooled human AB serum (Sigma, Bornem, Belgium) and 500 IU/ml interleukin (IL)-2 (R&D Systems, Minneapolis, MN, USA) and used as responder population in DC coculture experiments.
  • AIM-V medium Gibco BRL
  • human AB serum Sigma, Bornem, Belgium
  • 500 IU/ml interleukin (IL)-2 R&D Systems, Minneapolis, MN, USA
  • BM samples were aspirated by sternal puncture from hematologically normal patients undergoing cardiac surgery, after informed consent.
  • Peripheral blood mononuclear cells PBMC
  • Mononuclear cells were isolated by Ficoll-Hypaque gradient separation (LSM, ICN Biomedicals Inc., Costa Mesa, CA, USA).
  • the CD34 labeled cells were then sorted on a FACStar PLUS cell sorter (Becton Dickinson, Erembodegem, Belgium) equipped with an air-cooled argon ion laser ILT model 5500-A (Ion Laser Technology, Salt Lake City, UT, USA). Sort windows were set to include cells with low side scatter and with positive green fluorescence (CD34 + ). Purities of >95% were routinely obtained.
  • CD34 + cells were cultured in 2 ml of compete medium supplemented with 100 ng/ml granulocyte-macrophage colony-stimulating factor (GM-CSF; Leucomax, Novartis Pharma, Basel, Switzerland), 2.5 ng/ml tumor necrosis factor (TNF)- ⁇ (Roche Molecular Biochemicals, Mannheim, Germany) and 50 ng/ml stem cell factor (SCF; Biosource, Nivelle, Belgium) until day 5; afterwards, SCF was replaced by 1000 U/ml IL-4 (R&D Systems), which was added for the next 5-9 days. After 12 days of culture, a 15-20 fold total cell expansion was observed and cells exhibited typical markers of mature DC including CD1a, CD80, CD86 and HLA-DR (Fig. 3C).
  • GM-CSF granulocyte-macrophage colony-stimulating factor
  • TNF tumor necrosis factor
  • SCF stem cell factor
  • sorted CD34 + cells were first cultured for 8 days in complete medium containing 100 ng/ml IL-3, 100 ng/ml IL-6 and 50 ng/ml SCF (all from Biosource), followed by LC differentiation in GM-CSF (100 ng/ml) and IL-4 (1000 U/ml) for the next 4 weeks.
  • Immature monocyte-derived DC (iMo-DC) were generated from PBMC as described by Romani, N. et al., J. Exp. Med., 180:83-93 (1996). 14 Briefly, PBMC were allowed to adhere in AIM-V medium for 2 h at 37°C.
  • GM-CSF 100 ng/ml
  • IL-4 1000 U/ml
  • Maturation of iMo-DC was induced by adding 2.5 ng/ml TNF- ⁇ and 100 ng/ml lipopolysaccharide (LPS; Sigma) for 24 h starting from day 6 of the Mo-DC culture.
  • HLA-A2 subtyping was determined on BM-derived mononuclear cells and PBMC by indirect staining with the supernatant of the BB7-2 hybridoma (anti-HLA-A2; ATCC), followed by FITC-conjugated rabbit anti-mouse immunoglobulins (DAKO). HLA-A2 staining was analyzed by flow cytometry using a FACScan analytical flow cytometer (Becton Dickinson, Erembodegem, Belgium).
  • HLA-A*0201 restricted synthetic peptides were used: influenza virus peptide (M1; amino acids (aa) 58-66, GILGFVFTL); Melan-A peptide (MA; aa 27-35, AAGIGILTV).
  • M1 influenza virus peptide
  • MA Melan-A peptide
  • MA aa 27-35, AAGIGILTV
  • Peptides >95% pure were purchased from Sigma-Genosys (Cambridge, UK). Both peptides were dissolved in 100% DMSO to 10 mg/ml, further diluted to 1 mg/ml in serum-free IMDM and stored in aliquots at -70°C. Peptides were used at a final concentration of 20 mM.
  • T2 cells or HLA-A2 + iMo-DC were washed twice with IMDM and subsequently incubated with 20 ⁇ M peptide in serum-free-medium supplemented with 2.5 ⁇ g/ml ⁇ -2 microglobulin (Sigma) for 2 h at room temperature in 15-ml conical tubes. Afterwards, cells were washed twice and used as peptide controls in cytokine release assays.
  • pEGFP-N1 plasmid (CLONTECH Laboratories, Palo Alto, CA, USA) was used encoding an enhanced green fluorescent protein (EGFP) gene under the control of a CMV promoter, and pcDNA1.1/Melan-A containing the Melan-A/MART-1 gene driven by a CMV promoter (kindly provided by Dr. Pierre Van der Bruggen).
  • pcDNA1.1/Amp (Invitrogen, Carlsbad, CA, USA) was used as a backbone control vector.
  • Plasmids were propagated in E. Coli strain DH5 ⁇ (Gibco BRL) and purified on endotoxin-free QIAGEN®-tip 500 columns (Qiagen, Chatsworth, CA, USA).
  • plasmids were linearized, purified using a Genieprep kit (Ambion, Austin, TX, USA) and used as DNA templates for the in vitro transcription reaction.
  • pcDNA1.1/Melan-A was used as such for in vitro transcription under the control of a T7 promoter.
  • EGFP cDNA (a 0.8 kb HindIII-NotI fragment) was first subcloned into pcDNA1.1/Amp and subsequently cloned as a BamHI-XbaI fragment into pSP64 (Promega, Madison, WI, USA) that allows in vitro transcription under the control of an SP6 promoter.
  • K562 cells Prior to electroporation, K562 cells were washed twice with serum-free IMDM and resuspended to a final concentration of 5-10x10 6 cells/ml in Opti-MEM (Gibco BRL). After phenotypic analysis (performed in order to confirm the presence of CD1a + HLA-DR + DC in the cultures), 34-DC, 34-LC and Mo-DC were routinely harvested after respectively 12, 25 and 6 days of culture (unless stated otherwise), washed twice with serum-free IMDM, and resuspended to a final concentration of 10-40x10 6 cells/ml in Opti-MEM.
  • Lipofection of mRNA was performed using the cationic lipid DMRIE-C (Gibco BRL) according to manufacturer's instructions with minor modifications (Van Tendeloo, V.F.I. et al., Gene Ther., 5:700-707 (1998)). Briefly, K562 cells were washed twice with serum-free IMDM and resuspended to a final concentration of 1-2.10 6 cells/ml in Opti-MEM. 34-DC, 34-LC and Mo-DC were harvested after respectively 12, 25 and 6 days of culture, washed twice with serum-free IMDM, and resuspended to a final concentration of 1-2.10 6 cells/ml in Opti-MEM.
  • IVT mRNA Five ⁇ g of IVT mRNA, diluted in 250 ⁇ l Opti-MEM, was mixed with DMRIE-C, also diluted in 250 ⁇ l Opti-MEM, at a lipid:RNA ratio of 4:1. After 5-15 min of incubation at room temperature in order to allow RNA-lipid complexation, lipoplexes were added to the cells and allowed to incubate for 2 hours at 37°C. Alternatively, 5-20 ⁇ g of IVT mRNA was pulsed to the cells in the absence of DMRIE-C for 3-4 h at 37°C. Plasmid DNA lipofection was performed as described previously (Van Tendeloo, V.F.I. et al., Gene Ther., 5:700-707 (1998)). After lipofection or passive pulsing, fresh complete medium (including cytokines for DC) was added to each well.
  • EGFP-transfected cells were checked for EGFP expression 24-48 h after transfection by flow cytometric (FCM) analysis. Briefly, cells (1-5 x 10 5 ) were washed once in phosphate-buffered saline (PBS) supplemented with 1% FCS and resuspended in 0.5 ml of PBS supplemented with 1% BSA and 0.1% sodium azide. Ethidium bromide (EB) at a final concentration of 10 ⁇ g/ml was added directly prior to FCM analysis on a FACScan analytical flow cytometer (Becton Dickinson) to assess cell viability.
  • PBS phosphate-buffered saline
  • FCS phosphate-buffered saline
  • EB Ethidium bromide
  • gating was performed on cells exhibiting a large forward scatter (FSC) and side scatter (SSC) profile, i.e. DC, in order to allow exclusion of contaminating autologous lymphocytes. Gated DC were then evaluated for EGFP expression.
  • FSC forward scatter
  • SSC side scatter
  • Immunophenotyping was performed as described previously (Van Tendeloo, V.F.I. et al., Gene Ther., 5:700-707 (1998)).
  • the following monoclonal antibodies were used: CD1a-fluorescein isothiocyanate (FITC) (Ortho Diagnostic Systems, Beerse, Belgium), CD1a-phycoerythrin (PE) (Caltag Laboratories, San Francisco, CA, USA), CD14-PE, HLA-DR-PE, CD4-PE, CD80-PE (Becton Dickinson), CD40-FITC (BioSource, Zoersel, Belgium), CD86-PE (PharMingen, San Diego, CA, USA), CD13-FITC (DAKO) and CD83 (HB-15 clone; Immunotech, Marseille, France).
  • Nonreactive isotype-matched antibodies (Becton Dickinson) were used as controls.
  • 34-DC, 34-LC and iMo-DC were used as stimulator cells 24 h after transfection.
  • 6-day-cultured iMo-DC were allowed to mature for 24 h in the presence of TNF- ⁇ and LPS prior to transfection and used as stimulators 24 h after transfection.
  • iMo-DC were transfected with mRNA on day 6 of culture and, after 12-16 h to allow protein expression, TNF- ⁇ and LPS were added to induce final DC maturation. After an additional 24 h, mature transfected Mo-DC were used as stimulators.
  • iMo-DC pulsed with the Melan-A or an irrelevant influenza M1 peptide were used as stimulators.
  • Stimulators were washed twice and resuspended in AIM-V medium supplemented with 10% pooled human AB serum and 40 IU/ml IL-2. Responder CTL were washed vigorously 3-4 times and resuspended in AIM-V medium. Then, CTL (1 x 10 5 cells) were coincubated with stimulator cells (1x10 5 cells) in 96-round bottom plates for 24 h at 37°C in a total volume of 200 ⁇ l. Triplicate supernatant samples from these cocultures were tested for specific IFN- ⁇ secretion by an IFN- ⁇ ELISA (Biosource).
  • the background IFN- ⁇ secretion (defined as IFN- ⁇ released by the CTL exposed to unmodified DC) was subtracted from each of the observed measurements. Measurements are presented as IU/ml released by 10 5 responder cells/24 h.
  • mRNA electroporation at optimal settings showed a significantly reduced cell mortality rate as compared to cDNA electroporation at optimal settings (15% versus 51%, respectively).
  • DMRIE-C-mediated RNA and DNA lipofection showed a somewhat similar outcome in terms of efficiency and viability although optimal lipid:nucleic acid ratio (4:1 versus 3:1) as well as incubation time (2 h versus 6 h) varied for RNA and DNA lipofection, respectively (Table 1).
  • optimal lipid:nucleic acid ratio (4:1 versus 3:1) as well as incubation time (2 h versus 6 h) varied for RNA and DNA lipofection, respectively (Table 1).
  • RNA is extremely labile and has a short half-life time compared to DNA
  • Fig. 1B we also studied kinetics of EGFP expression following mRNA electroporation
  • Immature Mo-DC (iMo-DC) were generated from adherent PBMC in the presence of GM-CSF and IL-4. At day 5-6 of culture, Mo-DC were electroporated with EGFP mRNA. Optimization experiments revealed optimal settings similar to those of K562 cells (300 V, 150 ⁇ F), leading to maximal transfection efficiency combined with the lowest level of cell death. FCM analysis of EGFP expression showed more than 60% EGFP-expressing iMo-DC (Fig. 2A & Table 2). Mortality in Mo-DC after mRNA electroporation ranged from 15-30% (mean cell mortality rate 22 ⁇ 8%), although untransfected Mo-DC cultures already exhibited some degree of cell death (5-10%).
  • EGFP expression was analyzed by FCM to estimate transfection efficiency (% EGFP + DC).
  • iMo-DC immature Mo-DC
  • mMo-DC mature Mo-DC
  • 34-LC CD34 + progenitor-derived Langerhans cells
  • 34-DC CD34 + progenitor-derived dendritic cells.
  • SD standard deviation
  • Phenotype and maturation of mRNA-electroporated DC Phenotype and maturation of mRNA-electroporated DC:
  • mRNA-electroporated Mo-DC to differentiate to mature Mo-DC was evaluated by expression of mature DC markers including CD80 and CD83.
  • Fig. 3B shows that mRNA electroporation itself did not induce DC maturation, but that the maturation potential after electroporation was retained since mRNA-transfected immature Mo-DC were able to upregulate CD83 and CD80 in the presence of a maturation cocktail (TNF- ⁇ +LPS).
  • EGFP + 34-DC co-expressed HLA-DR, CD1a, CD80 and CD86. Similar findings were observed in 34-LC, with the exception that 34-LC exhibited lower levels of CD80 and CD86, compatible with their similarity to immature Langerhans-like DC (data not shown).
  • Mo-DC electroporation, lipofection or passive pulsing.
  • Mo-DC electroporated or lipofected with Melan-A mRNA markedly stimulated an HLA-A2 + Melan-A-specific CTL clone, as judged by IFN- ⁇ secretion (Fig. 4).
  • Mo-DC passively pulsed with Melan-A mRNA did not result in any CTL stimulation.
  • HLA-A2 + Mo-DC electroporated with EGFP mRNA or HLA-A2 - Mo-DC electroporated with Melan-A mRNA did not stimulate the CTL clone to produce IFN- ⁇ .
  • HLA-A2 + Mo-DC pulsed with the M1 influenza peptide did not elicit any specific IFN- ⁇ production.
  • iMo-DC immature Mo-DC
  • mMo-DC mature Mo-DC
  • 34-LC CD34 + progenitor-derived Langerhans cells
  • 34-DC CD34 + progenitor-derived DC
  • ⁇ BG IFN- ⁇ production below background.
  • Mo-DC obtained by culturing PBMC in the presence of GM-CSF and IL-4 for 5-7 days exhibit predominantly an immature phenotype (Romani, N. et al., J. Immunol. Methods, 196:137-151 (1996)). These immature Mo-DC are specialized in capturing large amounts of antigens from the environment (Sallusto, F., Lanzavecchia, A., J. Exp. Med., 179:1109-1118 (1994)). However, for optimal presentation to CTL, Mo-DC need to undergo a maturation process which can be induced by bacterial products (e.g. LPS), inflammatory cytokines (e.g.
  • LPS bacterial products
  • inflammatory cytokines e.g.
  • 34-LC and 34-DC can also be transfected by plasmid DNA electroporation (Van Tendeloo, V.F.I. et al., Gene Ther., 5:700-707 (1998)). Therefore, we evaluated whether plasmid DNA-transfected DC can also induce antigen-specific CTL activation.
  • HLA-A2 + 34-LC electoporated with plasmid DNA or IVT mRNA encoding Melan-A were incubated with the Melan-A specific CTL to evaluate IFN- ⁇ secretion (Fig. 6).
  • Example 2 EGFP RNA-transfection of immature monocyte-derived dendritic cells (generated from leukapheresis products and matured by a cocktail of IL-1 ⁇ + IL-6 + TNF ⁇ + PEG 2 under GMP conditions for clinical application) by electroporation
  • DC Dendritic Cells
  • the cell suspension were transferred in a 0,4-cm-gap electroporation-cuvette. Pulse were triggered at a voltage of 300 V and a capacitance of 150 ⁇ F with the Gene Pulser II (BioRad, Kunststoff, Germany) resulting in pulse time of 7-10 msec. Immediately after that the cell suspensions were transferred to 6-well-plates (1x10 6 DC/ well/3 ml culture medium supplemented with GM-CSF and IL-4). In the half number of the wells terminal maturation was induced by addition of IL-1 ⁇ , IL-6, TNF- ⁇ and PGE 2 as described (Feuerstein, B. et al., J. Immunol.
  • Example 3 EGFP RNA-transfection of monocyte-derived dendritic cells by electroporation - Titration of Voltage
  • DC Dendritic Cells
  • the Forward and Side Scatter analysis addition reveals that for monocyte-derived Dendritic Cells that are generated from leukapheresis products, RNA-transfected by electroporation, and fully matured by adding a maturation cocktail consisting of of IL-1 ⁇ , IL-6, TNF-a and PGE 2 (Feuerstein, B. et al., J. Immunol. Methods, 245: 15-29 (2000)) the use of 260 V is slightly better as the integrity of the cells is somewhat better preserved.
  • Immature DC (d6) - see Fig. 9A - were washed twice in RPMI and once in washing-solution of the OPTIMIX®-Kit (EQUIBIO, Maidstone Kent, U.K.). DC were adjusted to a final cell concentration of 10x10 6 /ml in OPTIMIX®--Medium. Then 0,200 ml of the cell suspension were mixed with or without 20 ⁇ g in vitro transcribed EGFP RNA in a 1,5 ml reaction tube. After incubation at room temperature for a maximum of 3 minutes the cell suspension was transferred in a 0,4-cm-gap electroporation-cuvette.
  • Pulses were triggered at the indicated voltage and a capacitance of 150 ⁇ F with the Gene Pulser II (BioRad, Kunststoff, Germany) resulting in pulse time of 7-10 msec.
  • the cell suspensions were transferred to 6-well-plates (1x10 6 DC/ well/3 ml culture medium). Terminal maturation was induced by addition of IL-1 ⁇ , IL-6, TNF-a and PGE 2 .
  • 48 h after electroporation the DC were counterstained with the indicated mouse mAbs and PE-conjugated anti-mouse Ig followed by FACS-analysis. The results are shown in Fig. 9B.
  • the phenotypic analysis reveals that for monocyte-derived Dendritic Cells that are generated from leukapheresis products, RNA-transfected by electroporation, and fully matured by adding a maturation cocktail consisting of of IL-1 ⁇ , IL-6, TNF-a and PGE 2 (Feuerstein, B. et al., J. Immunol. Methods, 245: 15-29 (2000)) the use of 260 V is slightly better as more cells are in the upper right quadrant, i.e. expressing both EGFP and the maturation markers CD83 and CD25.
  • Example 4 EGFP RNA-transfection of already matured monocyte-derived dendritic cells (generated from leukapheresis cells and matured by a cocktail of IL-1 ⁇ + IL-6 + TNF ⁇ + PEG 2 under GMP conditions for clinical application) by electroporation
  • DC Dendritic Cells
  • DC matured by TNF ⁇ + LPS are transfected only to a mean of 33 %, from the results depicted in Figs. 10A and H it can be concluded that mature monocyte-derived Dendritic Cells (DC) are efficiently transfected, and maintain EGFP expression over the 48h time period tested, if ... are matured by an optimised maturation cocktail consisting of IL-1 ⁇ + IL-6 + TNF ⁇ + PGE 2 .
  • DC Mature monocyte-derived Dendritic Cells
  • Example 5 mRNA-electroporated mature dendritic cells retain transgene expression, phenotypical properties and stimulatory capacity after cryopreservation
  • K562 cells were electroporated with EGFP mRNA and cryopreserved 3 or 24 hours after transfection.
  • K562 cells were resuspended in cryotubes (Nunc CryoTube Vials, Nalgene Nunc International, Denmark) at a concentration of 10 x 10 6 per mL in pure FCS.
  • the suspension was mixed on ice with an equal volume of FCS supplemented with 20% DMSO (Sigma, St. Louis, MO, USA).
  • Cell suspensions were slowly frozen (-1°C/min) to -80°C by using a cryo freezing container (Nalgene Nunc International). Cells were frozen at -80°C for more than 24 hours before use in further experiments.
  • Immature Mo-DC were electroporated with EGFP mRNA.
  • Cells were cryopreserved as immature DC 18 hours after transfection or as mature DC 24 hours after transfection. Maturation was induced by adding a maturation cocktail (TNF- ⁇ + PGE 2 + IL-1 + IL-6) directly after transfection.
  • FCM maturation cocktail

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Cell Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Microbiology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Mycology (AREA)
  • Epidemiology (AREA)
  • Biomedical Technology (AREA)
  • Zoology (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Wood Science & Technology (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Oncology (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Molecular Biology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Materials For Medical Uses (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
EP01115076A 2001-06-21 2001-06-21 Transfektion von eukaryotischen Zellen mit linearen Polynukleotiden mittels Elektroporation Withdrawn EP1270732A1 (de)

Priority Applications (12)

Application Number Priority Date Filing Date Title
EP01115076A EP1270732A1 (de) 2001-06-21 2001-06-21 Transfektion von eukaryotischen Zellen mit linearen Polynukleotiden mittels Elektroporation
CN028163621A CN1701121B (zh) 2001-06-21 2002-06-21 通过电穿孔用线性多核苷酸改善对真核细胞的转染
KR10-2003-7016741A KR20040025690A (ko) 2001-06-21 2002-06-21 진핵세포를 전기천공에 의하여 선형 폴리뉴크레오티드로형질감염시키는 개선된 형질감염 방법
PCT/EP2002/006897 WO2003000907A2 (en) 2001-06-21 2002-06-21 Improved transfection of eukaryotic cells with linear polynucleotides by electroporation
CA2451389A CA2451389C (en) 2001-06-21 2002-06-21 Improved transfection of eukaryotic cells with linear polynucleotides by electroporation
EP10185221.8A EP2308987B1 (de) 2001-06-21 2002-06-21 Transfektion von eukaryotischen Zellen mit linearen Polynukleotiden mittels Elektroporation
BR0210936-0A BR0210936A (pt) 2001-06-21 2002-06-21 Transfecção melhorada de células eucarióticas com polinucleotìdeos lineares por eletroporação
EP02748810.5A EP1397500B1 (de) 2001-06-21 2002-06-21 Verbesserte transfektion eukaryotischer zellen mit linearen polynukleotiden mittels elektroporation
JP2003507290A JP2004536598A (ja) 2001-06-21 2002-06-21 電気穿孔による真核細胞への線形ポリヌクレオチドの改良された形質移入
AU2008202507A AU2008202507B2 (en) 2001-06-21 2008-06-05 Improved transfection of eukaryotic cells with linear polynucleotides by electroporation
JP2010265545A JP2011050395A (ja) 2001-06-21 2010-11-29 電気穿孔による真核細胞への線形ポリヌクレオチドの改良された形質移入
AU2011201652A AU2011201652C1 (en) 2001-06-21 2011-04-12 Improved transfection of eukaryotic cells with linear polynucleotides by electroporation

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
EP01115076A EP1270732A1 (de) 2001-06-21 2001-06-21 Transfektion von eukaryotischen Zellen mit linearen Polynukleotiden mittels Elektroporation

Publications (1)

Publication Number Publication Date
EP1270732A1 true EP1270732A1 (de) 2003-01-02

Family

ID=8177780

Family Applications (3)

Application Number Title Priority Date Filing Date
EP01115076A Withdrawn EP1270732A1 (de) 2001-06-21 2001-06-21 Transfektion von eukaryotischen Zellen mit linearen Polynukleotiden mittels Elektroporation
EP10185221.8A Expired - Lifetime EP2308987B1 (de) 2001-06-21 2002-06-21 Transfektion von eukaryotischen Zellen mit linearen Polynukleotiden mittels Elektroporation
EP02748810.5A Expired - Lifetime EP1397500B1 (de) 2001-06-21 2002-06-21 Verbesserte transfektion eukaryotischer zellen mit linearen polynukleotiden mittels elektroporation

Family Applications After (2)

Application Number Title Priority Date Filing Date
EP10185221.8A Expired - Lifetime EP2308987B1 (de) 2001-06-21 2002-06-21 Transfektion von eukaryotischen Zellen mit linearen Polynukleotiden mittels Elektroporation
EP02748810.5A Expired - Lifetime EP1397500B1 (de) 2001-06-21 2002-06-21 Verbesserte transfektion eukaryotischer zellen mit linearen polynukleotiden mittels elektroporation

Country Status (8)

Country Link
EP (3) EP1270732A1 (de)
JP (2) JP2004536598A (de)
KR (1) KR20040025690A (de)
CN (1) CN1701121B (de)
AU (2) AU2008202507B2 (de)
BR (1) BR0210936A (de)
CA (1) CA2451389C (de)
WO (1) WO2003000907A2 (de)

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006072409A1 (de) * 2004-12-29 2006-07-13 JOHANNES GUTENBERG-UNIVERSITÄT MAINZ vertreten durch den Präsidenten Verfahren zur bestimmung der funktion von nukleinsäuresequenzen und den dadurch kodierten expressionsprodukten
EP1963514A2 (de) * 2005-12-13 2008-09-03 The Trustees Of The University Of Pennsylvania Verfahren zur phototransfikation von nukleinsäure in lebende zellen
EP2547360A1 (de) * 2010-03-15 2013-01-23 The Trustees of The University of Pennsylvania System und verfahren zur herstellung und lagerung aktivierter reifer dendritischer zellen
WO2014152211A1 (en) * 2013-03-14 2014-09-25 Moderna Therapeutics, Inc. Formulation and delivery of modified nucleoside, nucleotide, and nucleic acid compositions
US9095552B2 (en) 2012-04-02 2015-08-04 Moderna Therapeutics, Inc. Modified polynucleotides encoding copper metabolism (MURR1) domain containing 1
US9107886B2 (en) 2012-04-02 2015-08-18 Moderna Therapeutics, Inc. Modified polynucleotides encoding basic helix-loop-helix family member E41
US9157066B2 (en) 2005-12-13 2015-10-13 The Trustees Of The University Of Pennsylvania Transcriptome transfer produces cellular phenotype conversion
US9186372B2 (en) 2011-12-16 2015-11-17 Moderna Therapeutics, Inc. Split dose administration
US9828416B2 (en) 2012-04-02 2017-11-28 Modernatx, Inc. Modified polynucleotides for the production of secreted proteins
US10647960B2 (en) 2005-12-13 2020-05-12 The Trustees Of The University Of Pennsylvania Transcriptome transfer produces cellular phenotype conversion
WO2024008274A1 (en) * 2022-07-04 2024-01-11 Universiteit Antwerpen T regulatory cell modification

Families Citing this family (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2002322009A1 (en) 2001-05-18 2002-12-03 Mayo Foundation For Medical Education And Research Chimeric antigen-specific t cell-activating polypeptides
US20060222656A1 (en) 2005-04-01 2006-10-05 University Of Maryland, Baltimore MAGE-A3/HPV 16 peptide vaccines for head and neck cancer
US8513008B2 (en) 2004-10-07 2013-08-20 Argos Therapeutics, Inc. Mature dendritic cell compositions and methods for culturing same
AU2005294150B2 (en) 2004-10-07 2011-06-16 Coimmune, Inc. Mature dendritic cell compositions and methods for culturing same
ES2650569T3 (es) * 2005-04-08 2018-01-19 Argos Therapeutics, Inc. Composiciones de células dendríticas y métodos
DE102006005827B3 (de) * 2006-02-08 2007-07-26 Torzewski, Jan, Prof. Dr. med. mRNA-Transfektion von adulten Progenitorzellen zur spezifischen Gewebsregeneration
WO2013018778A1 (ja) * 2011-07-29 2013-02-07 独立行政法人理化学研究所 ウィルムス腫瘍遺伝子産物またはその断片をコードしている改変型核酸構築物を含んでいる免疫治療用細胞、当該細胞の製造方法、および当該核酸構築物
US10316332B2 (en) 2011-07-29 2019-06-11 Riken Cell for immunotherapy, including modified nucleic acid construct encoding Wilms tumor gene product
EA034110B1 (ru) * 2013-03-15 2019-12-27 Дзе Трастиз Оф Дзе Юниверсити Оф Пенсильвания Противораковые вакцины и способы лечения с их применением
CN104761644A (zh) * 2014-01-03 2015-07-08 百奇生物科技（苏州）有限公司 大肠杆菌表达的融合蛋白mbp-mart-1及其制备和应用
KR101518972B1 (ko) 2014-08-01 2015-05-18 제이더블유크레아젠 주식회사 수지상세포의 제조방법, 이에 의해 제조된 수지상세포 및 그 용도
CN104830794A (zh) * 2015-05-05 2015-08-12 杨光华 基于hpve7抗原的dc细胞、靶向性免疫细胞群及其制备方法和用途
CN112899308B (zh) * 2021-02-05 2022-03-11 北京鼎成肽源生物技术有限公司 一种电转染永生化树突状细胞的方法

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5554528A (en) * 1987-08-21 1996-09-10 Board Of Revents Of University Of Colorado Compositions and methods for inhibition of HIV production

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5124259A (en) * 1989-08-23 1992-06-23 Mitsui Toatsu Chemicals, Inc. Method for electroporation
GB9317380D0 (en) 1993-08-20 1993-10-06 Therexsys Ltd Transfection process
US6368855B1 (en) * 1996-06-11 2002-04-09 Antigen Express, Inc. MHC class II antigen presenting cells containing oligonucleotides which inhibit Ii protein expression
AU770952B2 (en) * 1999-03-01 2004-03-11 Genentech Inc. Antibodies for cancer therapy and diagnosis
PL354093A1 (en) * 1999-06-30 2003-12-29 Corixa Corporationcorixa Corporation Compositions and methods for the therapy and diagnosis of lung cancer

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5554528A (en) * 1987-08-21 1996-09-10 Board Of Revents Of University Of Colorado Compositions and methods for inhibition of HIV production

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
CELLA MARINA ET AL: "Origin, maturation and antigen presenting function of dendritic cells.", CURRENT OPINION IN IMMUNOLOGY, vol. 9, no. 1, 1997, pages 10 - 16, XP002166451, ISSN: 0952-7915 *
MORSE MICHAEL A ET AL: "Optimization of the sequence of antigen loading and CD40-ligand-induced maturation of dendritic cells.", CANCER RESEARCH, vol. 58, no. 14, 15 July 1998 (1998-07-15), pages 2965 - 2968, XP001037215, ISSN: 0008-5472 *
NAIR SMITA K ET AL: "Induction of primary carcinoembryonic antigen (CEA)-specific cytotoxic T lymphocytes in vitro using human dendritic cells transfected with RNA.", NATURE BIOTECHNOLOGY, vol. 16, no. 4, April 1998 (1998-04-01), pages 364 - 369, XP001026122, ISSN: 1087-0156 *
STROBEL I ET AL: "Human dendritic cells transfected with either RNA or DNA encoding influenza matrix protein M1 differ in their ability to stimulate cytotoxic T lymphocytes.", GENE THERAPY, vol. 7, no. 23, December 2000 (2000-12-01), pages 2028 - 2035, XP001034189, ISSN: 0969-7128 *
VAN TENDELOO VIGGO F I ET AL: "Highly efficient gene delivery by mRNA electroporation in human hematopoietic cells: Superiority to lipofection and passive pulsing of mRNA and to electroporation of plasmid cDNA for tumor antigen loading of dendritic cells.", BLOOD, vol. 98, no. 1, 1 July 2001 (2001-07-01), pages 49 - 56, XP001037914, ISSN: 0006-4971 *
ZHANG WEIPING ET AL: "Enhanced therapeutic efficacy of tumor RNA-pulsed dendritic cells after genetic modification with lymphotactin.", HUMAN GENE THERAPY, vol. 10, no. 7, 1 May 1999 (1999-05-01), pages 1151 - 1161, XP001037915, ISSN: 1043-0342 *

Cited By (27)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2005324086B2 (en) * 2004-12-29 2011-03-24 Biontech Ag Method for determining the function of nucleic acid sequences and expression products coded thereby
WO2006072409A1 (de) * 2004-12-29 2006-07-13 JOHANNES GUTENBERG-UNIVERSITÄT MAINZ vertreten durch den Präsidenten Verfahren zur bestimmung der funktion von nukleinsäuresequenzen und den dadurch kodierten expressionsprodukten
US10646590B2 (en) 2005-12-13 2020-05-12 The Trustees Of The University Of Pennsylvania Methods for phototransfecting nucleic acids into live cells
EP1963514A4 (de) * 2005-12-13 2010-05-19 Univ Pennsylvania Verfahren zur phototransfikation von nukleinsäure in lebende zellen
EP2602326A1 (de) * 2005-12-13 2013-06-12 The Trustees of the University of Pennsylvania Verfahren zur Phototransfikation von Nukleinsäure in lebende Zellen
US10883082B2 (en) 2005-12-13 2021-01-05 The Trustees Of The University Of Pennsylvania Transcriptome transfer produces cellular phenotype conversion
US10647960B2 (en) 2005-12-13 2020-05-12 The Trustees Of The University Of Pennsylvania Transcriptome transfer produces cellular phenotype conversion
US9845480B2 (en) 2005-12-13 2017-12-19 The Trustees Of The University Of Pennsylvania Transcriptome transfer produces cellular phenotype conversion
US10583204B2 (en) 2005-12-13 2020-03-10 The Trustees Of The University Of Pennsylvania Methods for transfecting nucleic acid into live cells
US9157066B2 (en) 2005-12-13 2015-10-13 The Trustees Of The University Of Pennsylvania Transcriptome transfer produces cellular phenotype conversion
EP1963514A2 (de) * 2005-12-13 2008-09-03 The Trustees Of The University Of Pennsylvania Verfahren zur phototransfikation von nukleinsäure in lebende zellen
EP3467112A3 (de) * 2005-12-13 2019-05-22 The Trustees of The University of Pennsylvania Verfahren zur transfektion von nukleinsäure in lebende zellen
EP2547360A1 (de) * 2010-03-15 2013-01-23 The Trustees of The University of Pennsylvania System und verfahren zur herstellung und lagerung aktivierter reifer dendritischer zellen
EP2547360A4 (de) * 2010-03-15 2013-11-20 Univ Pennsylvania System und verfahren zur herstellung und lagerung aktivierter reifer dendritischer zellen
US9186372B2 (en) 2011-12-16 2015-11-17 Moderna Therapeutics, Inc. Split dose administration
US9271996B2 (en) 2011-12-16 2016-03-01 Moderna Therapeutics, Inc. Formulation and delivery of PLGA microspheres
US9295689B2 (en) 2011-12-16 2016-03-29 Moderna Therapeutics, Inc. Formulation and delivery of PLGA microspheres
US9233141B2 (en) 2012-04-02 2016-01-12 Moderna Therapeutics, Inc. Modified polynucleotides for the production of proteins associated with blood and lymphatic disorders
US9828416B2 (en) 2012-04-02 2017-11-28 Modernatx, Inc. Modified polynucleotides for the production of secreted proteins
US9220792B2 (en) 2012-04-02 2015-12-29 Moderna Therapeutics, Inc. Modified polynucleotides encoding aquaporin-5
US9192651B2 (en) 2012-04-02 2015-11-24 Moderna Therapeutics, Inc. Modified polynucleotides for the production of secreted proteins
US9114113B2 (en) 2012-04-02 2015-08-25 Moderna Therapeutics, Inc. Modified polynucleotides encoding citeD4
US9107886B2 (en) 2012-04-02 2015-08-18 Moderna Therapeutics, Inc. Modified polynucleotides encoding basic helix-loop-helix family member E41
US9095552B2 (en) 2012-04-02 2015-08-04 Moderna Therapeutics, Inc. Modified polynucleotides encoding copper metabolism (MURR1) domain containing 1
US10258698B2 (en) 2013-03-14 2019-04-16 Modernatx, Inc. Formulation and delivery of modified nucleoside, nucleotide, and nucleic acid compositions
WO2014152211A1 (en) * 2013-03-14 2014-09-25 Moderna Therapeutics, Inc. Formulation and delivery of modified nucleoside, nucleotide, and nucleic acid compositions
WO2024008274A1 (en) * 2022-07-04 2024-01-11 Universiteit Antwerpen T regulatory cell modification

Also Published As

Publication number Publication date
AU2011201652A1 (en) 2011-04-28
CA2451389A1 (en) 2003-01-03
WO2003000907A3 (en) 2003-09-25
BR0210936A (pt) 2004-06-08
AU2008202507B2 (en) 2011-02-17
EP1397500B1 (de) 2014-09-17
CA2451389C (en) 2012-12-11
AU2011201652C1 (en) 2013-03-14
CN1701121B (zh) 2013-12-04
AU2008202507A1 (en) 2008-06-26
EP2308987A3 (de) 2011-04-27
JP2004536598A (ja) 2004-12-09
AU2011201652B2 (en) 2012-08-16
EP2308987A2 (de) 2011-04-13
EP2308987B1 (de) 2014-09-10
AU2011201652B8 (en) 2012-09-20
CN1701121A (zh) 2005-11-23
WO2003000907A2 (en) 2003-01-03
JP2011050395A (ja) 2011-03-17
EP1397500A2 (de) 2004-03-17
KR20040025690A (ko) 2004-03-24

Similar Documents

Publication Publication Date Title
US7547551B2 (en) Transfection of eukaryontic cells with linear polynucleotides by electroporation
AU2011201652B8 (en) Improved transfection of eukaryotic cells with linear polynucleotides by electroporation
Van Tendeloo et al. Highly efficient gene delivery by mRNA electroporation in human hematopoietic cells: superiority to lipofection and passive pulsing of mRNA and to electroporation of plasmid cDNA for tumor antigen loading of dendritic cells
Weissman et al. HIV gag mRNA transfection of dendritic cells (DC) delivers encoded antigen to MHC class I and II molecules, causes DC maturation, and induces a potent human in vitro primary immune response
Nair et al. Induction of primary carcinoembryonic antigen (CEA)-specific cytotoxic T lymphocytes in vitro using human dendritic cells transfected with RNA
Tuting et al. Autologous human monocyte-derived dendritic cells genetically modified to express melanoma antigens elicit primary cytotoxic T cell responses in vitro: enhancement by cotransfection of genes encoding the Th1-biasing cytokines IL-12 and IFN-α
EP1012240B1 (de) Krebs immuntherapie mit semi-allogenen zellen
JP2017533702A (ja) 生体分子の免疫細胞への送達
Bubenik Genetically engineered dendritic cell-based cancer vaccines
Tuyaerts et al. Induction of Influenza Matrix Protein 1 and MelanA-specific T lymphocytes in vitro using mRNA-electroporated dendritic cells
CA2367590A1 (en) In vitro induction of antigen-specific t-cells using dendritic cell-tumor cell or dendritic cell-viral cell derived immunogens
Philip et al. Dendritic cells loaded with MART-1 peptide or infected with adenoviral construct are functionally equivalent in the induction of tumor-specific cytotoxic T lymphocyte responses in patients with melanoma
Koido et al. Dendritic/tumor fusion cell-based vaccination against cancer
EP2302038A1 (de) Verwendung allogener Zelllinien zur Beladung von antigen-präsentierenden Zellen zur Auslösung oder Unterdrückung von Immunantworten
Met et al. Studies on mRNA electroporation of immature and mature dendritic cells: effects on their immunogenic potential
EP4089170A1 (de) Zusammensetzung, die antigen-präsentierende zellen umfasst, die mhc und tumorantigen gemeinsam exprimieren, und krebsbehandlung unter verwendung davon
Artusio et al. Transfection of human monocyte-derived dendritic cells with native tumor DNA induces antigen-specific T-cell responses in vitro.
Berlyn et al. Developing dendritic cell polynucleotide vaccination for prostate cancer immunotherapy
Tüting et al. DNA vaccines targeting dendritic cells for the immunotherapy of cancer
AU2002319256A1 (en) Improved transfection of eukaryotic cells with linear polynucleotides by electroporation
Markowicz et al. Nonviral transfection of human umbilical cord blood dendritic cells is feasible, but the yield of dendritic cells with transgene expression limits the application of this method in cancer immunotherapy
Ponsaerts Dendritic cell vaccines for immunity and tolerance: mRNA electroporation as a tool for efficient gene transfer
US6759036B1 (en) Fibrocyte-base vaccine formulations
O-SULLIVAN et al. Therapeutic properties of DNA-based fibroblast and dendritic cell vaccines in mice with squamous carcinoma
Morse et al. Dendritic cell-based approaches to cancer immunotherapy

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LI LU MC NL PT SE TR

AX Request for extension of the european patent

Free format text: AL;LT;LV;MK;RO;SI

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION HAS BEEN WITHDRAWN

18W Application withdrawn

Effective date: 20030206